Self-assembly label-free enzyme-free electrochemical HPV16 E7 detection platform as well as preparation method and application thereof

Abstract

The invention provides a self-assembly label-free enzyme-free electrochemical HPV16E7 detection platform as well as a preparation method and application thereof, and belongs to the technical field of biological analysis. According to the invention, a magnetic alpha-Fe2O3 / Fe3O4 (at) Au / Sub / BSA signal transmission medium, a CRISPR / Cas14a system and a double-foot DNA walker are organically combined to prepare a self-assembly label-free enzyme-free electrochemical HPV16E7 detection platform, and a more reliable and accurate means is provided for HPV16E7 detection; the linear detection range of the electrochemical HPV16E7 detection platform is 100 fg.mL <-1 >-10 [mu] g.mL <-1 >, the detection limit is 67.17 fg.mL <-1 >, and the quantification limit is 0.22 pg.mL <-1 >; the electrochemical HPV16E7 detection platform has the advantages of no need of enzyme, no need of marking, no need of pretreatment, fast and convenient detection process, low cost and good practicability.

Description

Technical Field

[0001] The present invention belongs to the field of bioanalysis technology, and specifically relates to a self-assembled label-free and enzyme-free electrochemical HPV16 E7 detection platform, a preparation method and an application thereof. Background Art

[0002] Cervical cancer refers to a malignant tumor that develops in the vaginal area of ​​the cervix and endocervical canal. It is the fourth most common cancer. Early cervical cancer can cause symptoms such as vaginal bleeding and discharge. If diagnosed promptly, it can be cured with medication or surgery. Its occurrence is closely related to infection with human papillomavirus (HPV), particularly HPV16, which is the essential cause of cervical cancer. Among them, the viral oncoprotein E7 (HPV16 E7) from HPV16 is responsible for cervical cancer cell proliferation and plays a key role in cell transformation and tumor progression. Therefore, detecting the presence of HPV16 E7 oncoprotein can be used for the early diagnosis of cervical cancer.

[0003] Currently, the main methods used for HPV16 E7 detection include polymerase chain reaction (PCR), real-time quantitative PCR (qPCR), RNA in situ hybridization (RNA-ISH), enzyme-linked immunosorbent assay (ELISA), immunohistochemistry (IHC), oncoprotein sequencing, and liquid-based cytology (LBC). However, these methods have limitations. For example, PCR and qPCR require specialized personnel for operation and analysis, resulting in high technical difficulty. RNA-ISH experiments are complex and time-consuming, potentially impacting clinical decision-making, and require experienced pathologists for analysis. Although ELISA can detect E7 antibody levels, its quantitative accuracy is limited. IHC has high sample requirements and may not detect E7 in early infection or in cases of low E7 expression. High-throughput sequencing is relatively expensive, limiting its use in routine testing. LBC results require the combination of other detection methods for accurate interpretation. To address the limitations of existing methods, it is particularly important to develop a high-sensitivity, high-specificity, low-cost, and rapid technology for early-stage HPV16 E7 oncoprotein detection. In contrast, electrochemical biosensors have recently become an important tool for biomolecular detection due to their rapidity, portability, and sensitivity.

[0004] As an emerging oncoprotein editing tool, CRISPR has achieved tremendous breakthroughs in the field of oncoprotein editing, but its application in detection is still in its infancy. Currently, there is relatively little research on electrochemical detection using CRISPR / Cas14a technology. Although detection strategies combining electrochemical methods with CRISPR / Cas14a technology can achieve highly sensitive and specific molecular diagnosis and have the advantages of being fast and simple, they suffer from problems such as unstable signal amplification and complex operation, making them difficult to use in actual detection.

[0005] Therefore, it is necessary to develop an electrochemical detection platform that can effectively improve the sensitivity and accuracy of HPV16 E7 detection. Summary of the Invention

[0006] In response to some of the shortcomings of the existing technology, the present invention provides a self-assembled, label-free, enzyme-free electrochemical HPV16 E7 detection platform, its preparation method, and application. The present invention organically combines a magnetic α-Fe2O3 / Fe3O4@Au / Sub / BSA signal transmission medium, a CRISPR / Cas14a system, and a bipedal DNA walker to prepare a self-assembled, label-free, enzyme-free electrochemical HPV16 E7 detection platform, providing a more reliable and accurate means for HPV16 E7 detection. The linear detection range of the electrochemical HPV16 E7 detection platform is 100 fg·mL -1 ~10 μg·mL -1 The detection limit was 67.17 fg·mL -1 The limit of quantification was 0.22 pg·mL -1 The electrochemical HPV16 E7 detection platform does not require enzymes, labels, or pretreatment, and the detection process is fast, convenient, low-cost, and has good practicality.

[0007] In order to achieve the above technical objectives, the present invention adopts the following technical means:

[0008] The present invention first provides a self-assembled label-free and enzyme-free electrochemical HPV16 E7 detection platform, which includes: an α-Fe2O3 / Fe3O4@Au / Sub / BSA signal transmission medium, a CRISPR / Cas14a signal amplification system, and an enzyme-free bipedal DNA walker;

[0009] The magnetic α-Fe2O3 / Fe3O4@Au / Sub / BSA signal transmission medium includes α-Fe2O3 / Fe3O4@Au nanoparticles, a hairpin structure reporter Sub assembled on the α-Fe2O3 / Fe3O4@Au nanoparticles, and a non-specific site blocker bovine serum albumin BSA;

[0010] The CRISPR / Cas14a signal amplification system is assembled from Cas12a enzyme and sgRNA;

[0011] The enzyme-free bipedal DNA walker includes HPV16 E7, aptamer A1, Mg 2+ , ssDNA D1 and ssDNA D2 and start the walking program.

[0012] Preferably, the nucleotide sequence of the sgRNA is shown as SEQ ID No. 1;

[0013] The nucleotide sequence of the hairpin structure reporter Sub is shown in SEQ ID No. 2;

[0014] The nucleotide sequences of the aptamer A1, single-stranded DNA D1 and single-stranded DNA D2 are shown in SEQ ID No. 3 to SEQ ID No. 5.

[0015] The present invention also provides a method for preparing the self-assembled label-free and enzyme-free electrochemical HPV16 E7 detection platform, the preparation method comprising:

[0016] (1) Construction of magnetic α-Fe2O3 / Fe3O4@Au / Sub / BSA signal transmission medium:

[0017] The α-Fe2O3 / Fe3O4@Au nanoparticle suspension was mixed with the hairpin structure reporter Sub, and then bovine serum albumin (BSA) was added to block the nonspecific adsorption sites to obtain the magnetic α-Fe2O3 / Fe3O4@Au / Sub / BSA signal transmission medium.

[0018] (2) Construction and activation of the enzyme-free bipedal DNA walker:

[0019] HPV16 E7 and aptamer A1 are mixed and incubated to obtain a binary complex HA. Single-stranded DNA D1 is then added to the binary complex HA. Complementary hybridization between single-stranded DNA D1 and aptamer A1 produces a ternary complex HAD. Single-stranded DNA D2 is then added to the complex to construct a DNA walker through complementary base pairing, resulting in a double-stranded bipedal DNA walker mixture with two single-stranded ends.

[0020] The double-stranded bipedal DNA walker mixture with two single-stranded ends was added to the magnetic α-Fe2O3 / Fe3O4@Au / Sub / BSA signal transmission medium, and Mg was added to it. 2+ The solution mixing reaction starts the DNA walker, and a mixture is obtained after the reaction is completed;

[0021] (3) Preparation of a self-assembled, label-free, enzyme-free electrochemical HPV16 E7 detection platform:

[0022] The Cas14a and sgRNA solution were incubated and assembled in HOLMES Buffer 1 to form a Cas14a-sgRNA binary complex; the Cas14a-sgRNA binary complex was added to the mixture obtained in step (2) for a cleavage reaction, and the Cas14a enzyme was inactivated after the reaction to obtain the self-assembled label-free enzyme-free electrochemical HPV16 E7 detection platform.

[0023] Preferably, in step (1), the method for preparing the α-Fe2O3 / Fe3O4@Au nanoparticles comprises:

[0024] S1. A mixed aqueous solution of FeCl3·6H2O and NH4H2PO4 was allowed to hydrothermally react to obtain an orange-red suspension, which was then centrifuged and washed to obtain α-Fe2O3 nanoparticles. The α-Fe2O3 was then partially reduced to Fe3O4 by calcination at high temperature using glucose as a reducing agent. After calcination, the mixture was ground to obtain magnetic α-Fe2O3 / Fe3O4 heterogeneous nanoparticles.

[0025] S2. The magnetic α-Fe2O3 / Fe3O4 heterogeneous nanoparticle suspension is ultrasonically dispersed, and then a polyethyleneimine (PEI) aqueous solution is added. After the reaction in a water bath, the solution is centrifuged, washed, and dried to obtain α-Fe2O3 / Fe3O4-PEI. Then, ice HAuCl4 solution is added to the α-Fe2O3 / Fe3O4-PEI suspension, and then sodium borohydride solution is added dropwise at a low temperature to reduce the solution to purple-red. The solution is centrifuged, washed, and dried to obtain a magnetic α-Fe2O3 / Fe3O4@Au nanocomposite.

[0026] Preferably, in step S1, in the mixed aqueous solution of FeCl3·6H2O and NH4H2PO4, the concentration of FeCl3 is 1mM to 60mM, and the concentration of NH4H2PO4 is 0.2mM to 20mM;

[0027] The conditions of the hydrothermal reaction are: reacting at 60-300° C. for 2-42 hours;

[0028] The mass ratio of glucose to α-Fe2O3 nanoparticles during the calcination process is 1:1 to 20:1;

[0029] The high temperature calcination condition is calcination at 300°C to 900°C for 0.2h to 15h;

[0030] In step S2, the mass ratio of the magnetic α-Fe2O3 / Fe3O4 heterogeneous nanoparticles, PEI, HAuCl4 and sodium borohydride is 1-10:50-300:1-40:20-200;

[0031] The conditions of the water bath reaction are: reaction at 70-100° C. for 0.5-10 hours;

[0032] The reduction reaction conditions are: reaction at 0°C for 5 to 60 minutes.

[0033] Preferably, in step (1), the α-Fe2O3 / Fe3O4@Au nanoparticle suspension and the hairpin structure reporter Sub are mixed in equal volumes, wherein the concentration of the α-Fe2O3 / Fe3O4@Au nanoparticle suspension is 1 mg·mL -1 ~40 mg mL -1 ;Sub concentration is 0.01μM~50μM;

[0034] The conditions for blocking the nonspecific adsorption sites with bovine serum albumin (BSA) are as follows: adding BSA with a mass concentration of 0.05% to 25% and incubating at 15 to 57° C. for 0.5 to 24 hours.

[0035] Preferably, the concentration of the α-Fe2O3 / Fe3O4@Au nanoparticle suspension is 10 mg·mL -1 , the concentration of Sub was 5 μM.

[0036] Preferably, in step (2), the concentration relationship of the aptamer A1, single-stranded DNA D1 and single-stranded DNA D2 in the system is 1 pM~10 mM: 1 pM~10 mM: 1 pM~10 mM; the volume ratio of HPV16 E7 to aptamer A1, single-stranded DNA D1 and single-stranded DNA D2 is 2:1:1:1;

[0037] The conditions for constructing the DNA walker are: reacting at 15-57° C. for 1-60 minutes.

[0038] Preferably, the concentrations of the aptamer A1, single-stranded DNA D1 and single-stranded DNA D2 in the system are all 1 μM.

[0039] Preferably, in step (2), the Mg 2+ The concentration is 0.1mM to 100mM, and the time of the mixing reaction initiated by the DNA walker is 0.1 to 3h.

[0040] Preferably, the Mg 2+ The concentration was 5 mM and the mixing reaction time was 1.5 h.

[0041] Preferably, in step (3), the Cas14a and sgRNA are mixed in equal volumes; wherein the concentration of Cas14a is 10nM to 500nM, and the concentration of sgRNA is 10nM to 500nM;

[0042] The incubation assembly conditions are: reacting at 15-57° C. for 1-60 min;

[0043] The cracking reaction is carried out for 10 to 90 minutes.

[0044] The conditions for the enzyme inactivation treatment are: reaction at 30-100° C. for 1-60 minutes.

[0045] Preferably, the concentrations of Cas14a and sgRNA are both 50 nM, and the optimal cleavage time is 30 min.

[0046] The present invention also provides the use of the self-assembled label-free and enzyme-free electrochemical HPV16 E7 detection platform in the preparation of an electrochemical sensor for detecting HPV16 E7 protein.

[0047] The present invention also provides an electrochemical sensor for detecting HPV16 E7 protein, wherein the electrochemical sensor uses the self-assembled label-free and enzyme-free electrochemical HPV16 E7 detection platform as a working electrode, an Ag / AgCl electrode and a Pt electrode as a reference and auxiliary electrode, respectively, and a [Fe(CN)6] 3- / 4- The solution was used as the electrolyte, and the HPV16 E7 protein concentration was detected by differential pulse voltammetry (DPV) using an electrochemical workstation.

[0048] Preferably, the concentration of KCl in the electrolyte is 0.1M, [Fe(CN)6] 3- / 4- The concentration of the solution is 5 mM;

[0049] During detection by the electrochemical nucleic acid sensor, the CV potential range is -0.1 V to +0.7 V, and the electrochemical impedance spectroscopy (EIS) is measured in the range of 0.1 Hz to 10 kHz with a signal amplitude of 0.1 V.

[0050] Compared with the prior art, the present invention has the following beneficial effects:

[0051] (1) During the preparation of the self-assembled, label-free, enzyme-free electrochemical HPV16 E7 detection platform described in the present invention, a high affinity exists between the target protein HPV16 E7 and the aptamer (A1). The 3'-end hairpin structure of A1 opens and binds to HPV16 E7 to form a binary complex HA. At the same time, the hairpin structure of single-stranded DNA D1 is specifically designed to partially complement the near-5' end sequence of A1, and hybridizes with HA to form a ternary complex (secondary structure free energy of -19.77 kcal / mol). When single-stranded DNA D2 is added to the ternary complex, due to the high degree of complementary pairing (19 nt) between single-stranded DNA D1 and single-stranded DNA D2, this structure promotes a reaction similar to DNA branch migration or strand displacement, ultimately forming a double-stranded bipedal DNA walker with two single-stranded ends (secondary structure free energy of -28.72 kcal / mol), thereby obtaining a structure with stronger binding stability. The displaced HA dissociates from the structure, triggering the aggregation of additional single-stranded DNA D1 and D2 molecules. This cyclical assembly generates more bipedal DNA walkers, achieving enzyme-free exponential signal amplification. The sub-reporter is then immobilized on the α-Fe2O3 / Fe3O4@Au nanocomposite via Au–S bonds and capped with BSA to form a nanobiosensor.

[0052] The constructed bipedal DNA walker was hybridized with the Sub reporter on the surface of α-Fe2O3 / Fe3O4@Au / Sub / BSA, and Mg 2+ The addition of triggers the action of a similar dependent cutting enzyme, causing the sub sequence to be cut from the hairpin structure into two single strands. Compared with the rigid double-stranded hairpin DNA, the flexible single-stranded DNA lies on the surface of the medium, thereby inhibiting the electron transfer behavior on the electrode surface and the current tends to decrease. At the same time, the released bipedal DNA walker continues to bind to the next cutting site as a free end and continuously releases the CRISPR / Cas14a activation sequence (Activator) to achieve signal amplification.

[0053] In the CRISPR / Cas14a-mediated cleavage reaction, the Cas14a-sgRNA ribonucleoprotein has no cutting activity in the unactivated state. When it recognizes the Activator present freely in the supernatant, the Activator binds to the sgRNA to form a stable R-loop structure, activating the non-specific ssDNA cutting activity (trans-cutting activity) of Cas14a. At this time, the Sub reporter fragments are further cut into negatively charged DNA fragments. Because the surface of the α-Fe2O3 / Fe3O4@Au nanocomposite is electropositive, the DNA fragments are adsorbed on the surface of the signal transmission medium through electrostatic adsorption, and the charge resistance is further increased, resulting in further closure of the electrical signal. In the absence of a target, the bipedal DNA walker cannot be formed smoothly, the Activator cannot be released, and the trans-cutting activity of Cas14a is silenced, making it impossible to cut the Sub reporter.

[0054] (2) The effective fixation of the signal transmission tool on the electrode surface is a key step in determining the performance of the electrochemical biosensor. The present invention prepares magnetically controllable α-Fe2O3 / Fe3O4 nanoparticles with large surface area and high surface energy. The α-Fe2O3 / Fe3O4 nanoparticles exhibit an appropriate saturation magnetization intensity, which allows the signal probe to be fixed on the MGCE surface through its magnetically induced self-assembly. In addition, due to its spherical structure with a large specific surface area and uniformity, it can evenly anchor more gold nanoparticles (AuNPs), thereby promoting the fixation of the thiol-modified reporter Sub on the α-Fe2O3 / Fe3O4@Au nanocomposite through Au-S bonds, laying a favorable foundation for the subsequent preparation of label-free self-assembled magnetic α-Fe2O3 / Fe3O4@Au / Sub / BSA signal transmission medium. In addition, the α-Fe2O3 / Fe3O4@Au nanocomposite can amplify the current signal. The α-Fe2O3 / Fe3O4@Au / Sub / BSA signal transmission medium described in the present invention is magnetic and can be quickly modified to the electrode surface through magnetic-induced self-assembly technology. The present invention utilizes the principle that the α-Fe2O3 / Fe3O4@Au / Sub / BSA signal transmission medium can be quickly removed by removing the magnetic core of the MGCE, and the electrode regeneration is simple and rapid; the required equipment requirements are not high, the operation is simple, and it is easy to carry.

[0055] (3) The present invention introduces nanomaterials to construct a three-dimensional DNA walker. Based on the existing technology, the bipedal structure DNA walker is introduced by increasing the number of these walker "legs", which makes up for the defects of the existing detection technology and achieves faster, longer-distance and more flexible movement. 2+After active cleavage of the hairpin reporter sub, the two feet are freed to continue cleaving the next site, thereby achieving enzyme-free exponential signal amplification and expansion. In addition, the cleaved ssDNA is designed to be complementary to the sgRNA base, which can act as an activator to activate the CRISPR / Cas system, achieving signal amplification and amplification functions in a single step without the need for PCR. This reduces background signal interference and enables better detection of the HPV16 E7 oncoprotein.

[0056] (4) After the present invention introduces the Cas14a-sgRNA binary complex and the Activator specific recognition combination, it will show strong nonspecific ssDNA trans-cleavage activity, cut the Sub reporter fragments, and quantitatively detect the target DNA by monitoring the changes in the electrochemical signal. In addition, the method of directly transferring the ribonucleoprotein (RNP) formed by Cas14a and sgRNA to the reporter surface during the preparation process has the advantages of being safer, faster, having lower off-target effects, and having higher detection efficiency.

[0057] (5) The HPV16 E7 oncoprotein detection platform prepared by the present invention does not require enzymes, labeling, or pretreatment; the detection process is fast and convenient, and the test results can be obtained in a few minutes. The amount required for each test is extremely small, and the cost is low; the reaction conditions are mild, the process is easy to control, and no operator training is required.

[0058] (6) The self-assembled, label-free, enzyme-free electrochemical HPV16 E7 detection platform of the present invention can detect the concentration of HPV16 E7 in a linear range of 100 fg mL -1 to 10 μg·mL -1 The detection limit was 67.17 fg·mL -1 (LOD = 3σ / m), limit of quantification: 0.22 pg·mL -1 (LOQ = 10σ / m), the detection limit is low and the detection range is wide.

[0059] The present invention also examined the analytical performance of the electrochemical detection platform under optimal conditions, using five independent electrodes to detect 10 ng·mL -1 The intra-day and inter-day reproducibility of the target HPV16 E7 oncoprotein were 1.90% and 1.86%, respectively, verifying that the electrochemical detection platform has good reproducibility. -1 interfering proteins and 10 ng·mL -1 The electrochemical detection platform was stored at 4 ° C for 30 days and detected 0.1 ng·mL -1 , 10ng·mL -1 and 1000 ng·mL-1 The current fluctuation range was within ±5% of the initial value, indicating good stability. Serum spiked samples with varying concentrations were also tested, with RSDs ranging from 1.79% to 4.72% and recoveries ranging from 98.46% to 115.78%, demonstrating the potential application of this electrochemical detection platform in clinical sample analysis. BRIEF DESCRIPTION OF THE DRAWINGS

[0060] Figure 1 This is a transmission electron microscope photo of α-Fe2O3 / Fe3O4@Au nanocomposite.

[0061] Figure 2 These are the cyclic voltammetry curves of each stage in the construction process of the enzyme-free label-free electrochemical detection platform under the conditions described in this example. In the figure, a is bare MGCE, b is MGCE / α-Fe2O3 / Fe3O4, c is MGCE / α-Fe2O3 / Fe3O4@Au, d is MGCE / α-Fe2O3 / Fe3O4@Au / Sub, e is MGCE / α-Fe2O3 / Fe3O4@Au / Sub / BSA, f is MGCE / α-Fe2O3 / Fe3O4@Au / Sub / BSA+DNA walker, and g is MGCE / α-Fe2O3 / Fe3O4@Au / Sub / BSA+DNA walker+Cas14a-sgRNA.

[0062] Figure 3 The current response of α-Fe2O3 / Fe3O4@Au / Sub / BSA magnetic nanosignal transmission medium to HPV16 E7 oncoprotein and human epidermal growth factor receptor 2 (HER2), plasmin, epithelial cell adhesion molecule (EpCAM); carcinoembryonic antigen (CEA) and their mixture (Mix).

[0063] Figure 4 The linear relationship between the DPV curve current and the logarithmic concentration of HPV16 E7 detected over a series of HPV16 E7 concentrations. DETAILED DESCRIPTION

[0064] The present invention will be further described below in conjunction with the accompanying drawings and specific examples, but the scope of protection of the present invention is not limited thereto. In the following examples, the technical means used are conventional means well known to those skilled in the art. Various processes and methods not described in detail are conventional methods well known in the art. The sources, trade names and components of the reagents used are indicated when they first appear, and the same reagents used thereafter are the same as those indicated for the first time unless otherwise specified; the reagents, materials, etc. involved are all obtained from commercial sources unless otherwise specified.

[0065] The Cas14a (Cas12f1) involved in the following examples was derived from Yizhi Biotechnology Co., Ltd., sgRNA was purchased from Ji'an Biotechnology Co., Ltd., HOLMES Buffer 1 was ordered from Tulu Biotechnology Co., Ltd., BSA was from Saiguo Biotechnology Co., Ltd., MgCl2 and SDS-PAGE loading buffer were purchased from Biyuntian Biotechnology Co., Ltd., PAGE color rapid gel preparation kit was purchased from Youlandi Biotechnology Co., Ltd., HPV16 E7 oncoprotein was purchased from Jinan Protein Technology Co., Ltd., and DNA sequences were purchased from Shanghai Shenggong Biological Co., Ltd. The nucleotide sequences involved are:

[0066] sgRNA:GGGCU UCACU GAUAA AGUGG AGAAC CGCUU CACCA AAAGC UGUCC CUUAGGGGAU UAGAA CUUGA GUGAA GGUGG GCUGC UUGCA UCAGC CUAAUGUCGA GAAGU GCUUU CUUCGGAAAG UAACC CUCGA AACAA AUUCA UUUUUCCUCU CCAAU UCUGC ACAAG AAAGU UGCAG AACCCGAAUA GACGA AUGAAGGAAU GCAAC UGGGU GCCAU ACACU UCACA (SEQ ID No. 1), replace u with t in the sequence listing.

[0067] Sub:SH-(CH2)6-TTTTT TTTTT TTTGG GTGGG TGGGT GGGTG GGTAA TGTGA AGTGTATGGC ACCCA(SEQ ID No.2)

[0068] A1:CCCTT CATCA TTAAC CCGTC CACGC GC(SEQ ID No.3)

[0069] D1:CGGTT AATGA TGAAA TTAAG CAATA CACGT CTCTT CTCCG AGCCG GTCGA AATAGTCACA TA (SEQ ID No. 4)

[0070] D2:GGCTT AATTT CATCA TTAAC TAATA CACGT CTCTT CTCCG AGCCG GTCGA AATAGTCACA TA(SEQ ID No.5)

[0071] Example 1: Preparation of α-Fe2O3 / Fe3O4@Au nanoparticles

[0072] (1) Weigh 12.42 mg NH4H2PO4 and 0.541 g FeCl3·6H2O and dissolve them in 100 mL deionized water. Then take 80 mL of the mixed solution and place it in a hydrothermal reactor at 2°C·min. -1 The hydrothermal reaction was carried out at 220°C at a heating rate of 24 hours. After the reaction, the solid product was removed, washed alternately with deionized water and anhydrous ethanol for 6 times, centrifuged, and dried to obtain the precursor α-Fe2O3 nanoparticles.

[0073] (2) Using glucose as a reducing agent, magnetic α-Fe2O3 / Fe3O4 heterogeneous nanoparticles were prepared by calcination reduction method. The mass ratio of glucose to α-Fe2O3 nanoparticles during the calcination process was 1:7. The specific process is as follows:

[0074] 0.081 g of precursor α-Fe2O3 nanoparticles and 0.5673 g of glucose were placed in a crucible and mixed evenly. The crucible was placed in a programmable temperature-controlled furnace and heated at 3 °C·min -1 The mixture was calcined at a heating rate of 400 °C for 1 h and ground to obtain magnetic α-Fe2O3 / Fe3O4 heterogeneous nanoparticles.

[0075] (3) 1.5069 g of polyethyleneimine (PEI) was dissolved in 150 mL of distilled water and ultrasonically dispersed for 30 min. Then, 75.4 mg of α-Fe2O3 / Fe3O4 heterogeneous nanoparticles were added thereto and ultrasonicated for 30 min. Then, the mixture was magnetically stirred in a 90 °C water bath for 1 h. After the reaction was completed, the mixture was cooled, magnetically separated, washed once with alcohol, washed twice with water, and dried to obtain the α-Fe2O3 / Fe3O4-PEI intermediate.

[0076] (4) Weigh 15 mg of magnetic α-Fe2O3 / Fe3O4-PEI intermediate and ultrasonically disperse it in 150 mL of double-distilled water to form a suspension. Then, 1 mL of a 20 mg·mL -1 The HAuCl4 solution was added dropwise to the suspension, ultrasonicated in an ice bath for 30 min, and then 9 mL of freshly prepared NaBH4 (0.075%) solution was slowly added. Stirring was continued in an ice bath for 15 min. The solution turned dark gray, and was magnetically separated and washed. After drying, α-Fe2O3 / Fe3O4@Au nanocomposites were obtained.

[0077] Figure 1This is a transmission electron micrograph of the α-Fe2O3 / Fe3O4@Au nanocomposite. The electron micrograph shows that AuNPs are evenly distributed on the surface of the α-Fe2O3 / Fe3O4 heterostructure nanoparticles. This uniform distribution laid a solid foundation for the subsequent construction of magnetic signal transmission media using Au-S. The average particle size of the AuNPs is approximately 8.14 nm, and the average particle size of the prepared nanoparticles is approximately 126.23 nm.

[0078] Example 2: Preparation of α-Fe2O3 / Fe3O4@Au / Sub / BSA signal transmission medium

[0079] (1) The α-Fe2O3 / Fe3O4@Au nanocomposite prepared in Example 1 was ultrasonically dispersed in ultrapure water to a concentration of 20 mg·mL -1 The α-Fe2O3 / Fe3O4@Au nanocomposite suspension is set aside.

[0080] Take 1OD of Sub, add TCEP (10mM) at a molar ratio of 1:100, and then adjust to 100μM with TE buffer. React at room temperature for 30 minutes to reduce disulfide bonds. Subsequently, dilute the Sub to a concentration of 10μM with TE buffer for use.

[0081] (2) Mix 15 μL of 10 μM Sub solution with 15 μL of 20 mg mL -1 The α-Fe2O3 / Fe3O4@Au nanocomplex was mixed and incubated at 4°C overnight (14 h), washed with PBS to remove unbound Sub, and magnetic separation was performed to remove the supernatant to obtain α-Fe2O3 / Fe3O4@Au / Sub.

[0082] (3) Use BSA to block the nonspecific adsorption sites of α-Fe2O3 / Fe3O4@Au / Sub: add 30 μL BSA (2.5 wt%) to α-Fe2O3 / Fe3O4@Au / Sub and incubate at room temperature for 0.5 h. Wash the unbound BSA with PBS, remove the supernatant by magnetic separation, and successfully construct the signal transmission medium α-Fe2O3 / Fe3O4@Au / Sub / BSA. Add 30 μL ultrapure water to disperse it to obtain α-Fe2O3 / Fe3O4@Au / Sub / BSA suspension.

[0083] 9 μL of α-Fe2O3 / Fe3O4@Au / Sub / BSA suspension and 9 μL of α-Fe2O3 / Fe3O4@Au / Sub suspension were added dropwise to the polished and cleaned MGCE surface and dried at room temperature to obtain MGCE / α-Fe2O3 / Fe3O4@Au / Sub and MGCE / α-Fe2O3 / Fe3O4@Au / Sub / BSA.

[0084] The MGCE / α-Fe2O3 / Fe3O4@Au / Sub / BSA was tested using DPV in the range of -0.05V to -0.5V, and the peak current was measured to be 46.49μA.

[0085] Example 3: Preparation and activation of a bipedal DNA walker

[0086] (1) 10 μL of a 10 ng·mL -1 HPV16 E7 was reacted with 5 μL of 1 μM A1 at 37°C for 45 minutes to form a binary complex HA; 5 μL of 1 μM single-stranded DNA D1 was added to the binary complex HA and reacted at 37°C for 45 minutes. A hairpin structure was designed in the single-stranded DNA D1, which was partially complementary to the near-5' end sequence of A1. After the reaction, a ternary complex HAD was formed; then 5 μL of 1 μM single-stranded DNA D2 was added to the ternary complex HAD and a chain displacement reaction was carried out at 37°C for 45 minutes. After the reaction, a double-stranded bipedal DNA walker mixture with two single-stranded ends was formed.

[0087] (2) The double-stranded bipedal DNA walker mixture with two single-stranded ends was added to the signal transmission medium α-Fe2O3 / Fe3O4@Au / Sub / BSA of Example 2, and then 5 μL of 2.5 mM Mg was added thereto. 2+ The solution was mixed and allowed to stand at room temperature for 1.5 hours, and the DNA walking program was started. During the DNA walking program, the Mg 2+ Dependent DNA enzyme, the Sub sequence was divided into two parts, and after the reaction was completed, magnetic separation was performed, the supernatant was discarded, and PBS was washed to obtain a mixture, which was recorded as α-Fe2O3 / Fe3O4@Au / Sub / BSA+DNA walker.

[0088] (3) 30 μL of ultrapure water was added to the α-Fe2O3 / Fe3O4@Au / Sub / BSA+DNA walker to obtain a suspension. 9 μL of the suspension was added dropwise to a polished, cleaned MGCE surface and dried at room temperature to obtain the MGCE / α-Fe2O3 / Fe3O4@Au / Sub / BSA+DNA walker. The walker was tested using DPV in the range of -0.05 V to -0.5 V, with a peak current of 20.56 μA.

[0089] Example 4: Assembly and activation of the label-free CRISPR / Cas14a system

[0090] (1) 5 μL of 100 nM Cas14a solution and 5 μL of 100 nM sgRNA solution were assembled in 10 μL of HOLMES Buffer 1 and incubated at room temperature for 20 min to form a Cas14a-sgRNA binary complex.

[0091] (2) Take 20 μL of the Cas14a-sgRNA binary complex and add it to 30 μL of the α-Fe2O3 / Fe3O4@Au / Sub / BSA+DNA walker obtained in Example 3, mix gently, and then lyse in the dark for 30 minutes. After the reaction, heat at 80°C for 10 minutes to inactivate the Cas14a enzyme, separate magnetically, and wash with PBS to obtain the self-assembled label-free enzyme-free electrochemical HPV16 E7 detection platform, which is recorded as α-Fe2O3 / Fe3O4@Au / Sub / BSA+DNA walker+Cas14a-sgRNA.

[0092] (3) Add 30 μL of ultrapure water to the α-Fe2O3 / Fe3O4@Au / Sub / BSA+DNA walker+Cas14a-sgRNA to disperse it and obtain a suspension. Take 9 μL of the suspension and drop it onto the clean MGCE surface and dry it at room temperature to obtain MGCE / α-Fe2O3 / Fe3O4@Au / Sub / BSA+DNA walker+Cas14a-sgRNA, completing the assembly and activation of the label-free CRISPR / Cas14a system.

[0093] Example 5: Preparation of a self-assembled, label-free, enzyme-free electrochemical HPV16 E7 detection platform

[0094] (1) Construction of magnetic α-Fe2O3 / Fe3O4@Au / Sub / BSA signal transmission medium:

[0095] S1. Ultrasonic dispersion of the α-Fe2O3 / Fe3O4@Au nanocomposite prepared in Example 1 in ultrapure water to a concentration of 30 mg mL -1 The α-Fe2O3 / Fe3O4@Au nanocomposite suspension is set aside.

[0096] Take 1OD of Sub, add TCEP (10mM) at a molar ratio of 1:100, and then adjust to 100μM with TE buffer. React at room temperature for 30 minutes to reduce disulfide bonds. Subsequently, dilute the Sub to a concentration of 10μM with TE buffer for use.

[0097] S2. Mix 15 μL of 20 μM Sub solution with 15 μL of 30 mg mL -1The α-Fe2O3 / Fe3O4@Au nanocomplex was mixed and incubated at 4°C overnight (14 h), washed with PBS to remove unbound Sub, and magnetic separation was performed to remove the supernatant to obtain α-Fe2O3 / Fe3O4@Au / Sub.

[0098] S3. Use BSA to block the nonspecific adsorption sites of α-Fe2O3 / Fe3O4@Au / Sub: add 30 μL BSA (0.25 wt%) to α-Fe2O3 / Fe3O4@Au / Sub and incubate at room temperature for 0.5 h. Wash the unbound BSA with PBS, remove the supernatant by magnetic separation, and construct the signal transmission medium α-Fe2O3 / Fe3O4@Au / Sub / BSA.

[0099] (2) Construction and activation of the enzyme-free bipedal DNA walker:

[0100] S1. Add 10 μL of 10 ng·mL -1 HPV16 E7 was reacted with 5 μL of 1 μM A1 at 37°C for 45 minutes to form a binary complex HA; 5 μL of 1 μM single-stranded DNA D1 was added to the binary complex HA and reacted at 37°C for 45 minutes. After the reaction, a ternary complex HAD was formed; then 5 μL of 1 μM single-stranded DNA D2 was added to the ternary complex HAD and a chain displacement reaction was carried out at 37°C for 45 minutes. After the reaction, a double-stranded bipedal DNA walker mixture with two single-stranded ends was formed.

[0101] S2. Add 25 μL of the double-stranded bipedal DNA walker mixture with two single-stranded ends to the signal transmission medium α-Fe2O3 / Fe3O4@Au / Sub / BSA obtained in step (1), and then add 5 μL of 5 mM Mg 2+ The solution was mixed, allowed to stand at room temperature for 1.5 h, and the DNA walking program was started to obtain an enzyme-free bipedal DNA walker.

[0102] (3) Preparation of a self-assembled, label-free, enzyme-free electrochemical HPV16 E7 detection platform:

[0103] S1. Assemble 5 μL of 50 nM Cas14a solution and 5 μL of 50 nM sgRNA solution in 10 μL of HOLMES Buffer 1 and incubate at room temperature for 20 min to form a Cas14a-sgRNA binary complex.

[0104] S2. Take 20 μL of the Cas14a-sgRNA binary complex and add it to 30 μL of the α-Fe2O3 / Fe3O4@Au / Sub / BSA+DNA walker obtained in Example 3, mix gently, and then cleave in the dark for 10 minutes. After the reaction, heat at 80°C for 10 minutes to inactivate the Cas14a enzyme, separate magnetically, and wash with PBS to obtain the self-assembled label-free and enzyme-free electrochemical HPV16 E7 detection platform.

[0105] 30 μL of ultrapure water was added to the self-assembled, label-free, enzyme-free electrochemical HPV16 E7 detection platform to disperse the suspension. 9 μL of the suspension was dropwise applied to a clean MGCE surface and dried at room temperature to obtain the MGCE / α-Fe2O3 / Fe3O4@Au / Sub / BSA+DNA walker+Cas14a-sgRNA. This was tested using DPV at -0.05V to -0.5V, with a peak current of 25.56 μA.

[0106] Example 6: Preparation of a self-assembled, label-free, enzyme-free electrochemical HPV16 E7 detection platform

[0107] (1) Construction of magnetic α-Fe2O3 / Fe3O4@Au / Sub / BSA signal transmission medium:

[0108] S1. Ultrasonic dispersion of the α-Fe2O3 / Fe3O4@Au nanocomposite prepared in Example 1 in ultrapure water to a concentration of 20 mg·mL -1 The α-Fe2O3 / Fe3O4@Au nanocomposite suspension is set aside.

[0109] Take 1OD of Sub, add TCEP (10mM) at a molar ratio of 1:100, and then adjust to 100μM with TE buffer. React at room temperature for 30 minutes to reduce disulfide bonds. Subsequently, dilute the Sub to a concentration of 10μM with TE buffer for use.

[0110] S2. Mix 15 μL of 10 μM Sub solution with 15 μL of 20 mg mL -1 The α-Fe2O3 / Fe3O4@Au nanocomplex was mixed and incubated at 4°C overnight (14 h), washed with PBS to remove unbound Sub, and magnetic separation was performed to remove the supernatant to obtain α-Fe2O3 / Fe3O4@Au / Sub.

[0111] S3. Use BSA to block the nonspecific adsorption sites of α-Fe2O3 / Fe3O4@Au / Sub: add 30μL BSA (5.0wt%) to α-Fe2O3 / Fe3O4@Au / Sub and incubate at room temperature for 0.5h. Wash the unbound BSA with PBS, remove the supernatant by magnetic separation, and construct the signal transmission medium α-Fe2O3 / Fe3O4@Au / Sub / BSA.

[0112] (2) Construction and activation of the enzyme-free bipedal DNA walker:

[0113] S1. Add 10 μL of 10 ng·mL -1 HPV16 E7 was reacted with 5 μL of 10 μM A1 at 37°C for 30 minutes to form a binary complex HA; 5 μL of 10 μM single-stranded DNA D1 was added to the binary complex HA and reacted at 37°C for 45 minutes. After the reaction, a ternary complex HAD was formed; then 5 μL of 10 μM single-stranded DNA D2 was added to the ternary complex HAD and a chain displacement reaction was carried out at 37°C for 30 minutes. After the reaction, a double-stranded bipedal DNA walker mixture with two single-stranded ends was formed.

[0114] S2. Add 25 μL of the double-stranded bipedal DNA walker mixture with two single-stranded ends to the signal transmission medium α-Fe2O3 / Fe3O4@Au / Sub / BSA obtained in step (1), and then add 5 μL of 10 mM Mg 2+ The solution was mixed, allowed to stand at room temperature for 2.5 h, and the DNA walking program was started to obtain an enzyme-free bipedal DNA walker.

[0115] (3) Preparation of a self-assembled, label-free, enzyme-free electrochemical HPV16 E7 detection platform:

[0116] S1. Assemble 5 μL of 100 nM Cas14a solution and 5 μL of 100 nM sgRNA solution in 10 μL of HOLMES Buffer 1 and incubate at room temperature for 20 min to form a Cas14a-sgRNA binary complex.

[0117] S2. Take 20 μL of the Cas14a-sgRNA binary complex and add it to 30 μL of the α-Fe2O3 / Fe3O4@Au / Sub / BSA+DNA walker obtained in Example 3, mix gently, and then cleave in the dark for 20 minutes. After the reaction, heat at 95°C for 5 minutes to inactivate the Cas14a enzyme, magnetically separate, and wash with PBS to obtain the self-assembled label-free and enzyme-free electrochemical HPV16 E7 detection platform.

[0118] 30 μL of ultrapure water was added to the self-assembled, label-free, enzyme-free electrochemical HPV16 E7 detection platform to disperse the suspension. 9 μL of the suspension was dropwise applied to a clean MGCE surface and dried at room temperature to obtain the MGCE / α-Fe2O3 / Fe3O4@Au / Sub / BSA+DNA walker+Cas14a-sgRNA. This was tested using DPV at -0.05V to -0.5V, with a peak current of 17.28 μA.

[0119] Example 7: Preparation of a self-assembled, label-free, enzyme-free electrochemical HPV16 E7 detection platform

[0120] (1) Construction of magnetic α-Fe2O3 / Fe3O4@Au / Sub / BSA signal transmission medium:

[0121] S1. Ultrasonic dispersion of the α-Fe2O3 / Fe3O4@Au nanocomposite prepared in Example 1 in ultrapure water to a concentration of 20 mg·mL -1 The α-Fe2O3 / Fe3O4@Au nanocomposite suspension is set aside.

[0122] Take 1OD of Sub, add TCEP (10mM) at a molar ratio of 1:100, and then adjust to 100μM with TE buffer. React at room temperature for 30 minutes to reduce disulfide bonds. Subsequently, dilute the Sub to a concentration of 10μM with TE buffer for use.

[0123] S2. Mix 15 μL of 10 μM Sub solution with 15 μL of 20 mg mL -1 The α-Fe2O3 / Fe3O4@Au nanocomplex was mixed and incubated at 4°C overnight (14 h), washed with PBS to remove unbound Sub, and magnetic separation was performed to remove the supernatant to obtain α-Fe2O3 / Fe3O4@Au / Sub.

[0124] S3. Use BSA to block the nonspecific adsorption sites of α-Fe2O3 / Fe3O4@Au / Sub: add 30 μL BSA (2.5wt%) to α-Fe2O3 / Fe3O4@Au / Sub and incubate at room temperature for 0.5 h. Wash the unbound BSA with PBS, remove the supernatant by magnetic separation, and construct the signal transmission medium α-Fe2O3 / Fe3O4@Au / Sub / BSA.

[0125] (2) Construction and activation of the enzyme-free bipedal DNA walker:

[0126] S1. Add 10 μL of 10 ng·mL -1HPV16 E7 was reacted with 5 μL of 10 nM A1 at 37°C for 40 min to form a binary complex HA; 5 μL of 10 nM single-stranded DNA D1 was added to the binary complex HA and reacted at 37°C for 40 min, after which a ternary complex HAD was formed; then 5 μL of 10 nM single-stranded DNA D2 was added to the ternary complex HAD and a chain displacement reaction was carried out at 37°C for 40 min, after which a double-stranded bipedal DNA walker mixture with two single-stranded ends was formed.

[0127] S2. Add 25 μL of the double-stranded bipedal DNA walker mixture with two single-stranded ends to the signal transmission medium α-Fe2O3 / Fe3O4@Au / Sub / BSA obtained in step (1), and then add 5 μL of 25 mM Mg 2+ The solution was mixed, allowed to stand at room temperature for 1 h, and the DNA walking program was started to obtain an enzyme-free bipedal DNA walker.

[0128] (3) Preparation of a self-assembled, label-free, enzyme-free electrochemical HPV16 E7 detection platform:

[0129] S1. Assemble 5 μL of 100 nM Cas14a solution and 5 μL of 200 nM sgRNA solution in 10 μL of HOLMES Buffer 1 and incubate at room temperature for 10 min to form a Cas14a-sgRNA binary complex.

[0130] S2. Take 20 μL of the Cas14a-sgRNA binary complex and add it to 30 μL of the α-Fe2O3 / Fe3O4@Au / Sub / BSA+DNA walker obtained in Example 3, mix gently, and then cleave and react in the dark for 60 minutes. After the reaction, heat at 80°C for 10 minutes to inactivate the Cas14a enzyme, magnetically separate, and wash with PBS to obtain the self-assembled label-free and enzyme-free electrochemical HPV16 E7 detection platform.

[0131] 30 μL of ultrapure water was added to the self-assembled, label-free, enzyme-free electrochemical HPV16 E7 detection platform to disperse the suspension. 9 μL of the suspension was dropwise applied to a clean MGCE surface and dried at room temperature to obtain the MGCE / α-Fe2O3 / Fe3O4@Au / Sub / BSA+DNA walker+Cas14a-sgRNA. This was tested using DPV at -0.05V to -0.5V, yielding a peak current of 14.88 μA.

[0132] Example 8: Construction of an electrochemical sensor containing an enzyme-free label-free electrochemical HPV16 E7 detection platform

[0133] In this example, cyclic voltammetry was used to investigate the construction process of an electrochemical sensor containing an enzyme-free label-free electrochemical HPV16 E7 detection platform. The specific experimental steps are as follows.

[0134] Preparation of MGCE / α-Fe2O3 / Fe3O4: α-Fe2O3 / Fe3O4 heterogeneous nanoparticles were ultrasonically dispersed in ultrapure water to a concentration of 10 mg mL -1 α-Fe2O3 / Fe3O4 heterogeneous nanoparticle suspension was prepared, and then 9 μL of the suspension was dropped onto the cleaned MGCE surface and dried at room temperature to obtain the MGCE / α-Fe2O3 / Fe3O4 modified electrode.

[0135] Preparation of MGCE / α-Fe2O3 / Fe3O4@Au: The α-Fe2O3 / Fe3O4@Au nanocomposite prepared in Example 1 was ultrasonically dispersed in ultrapure water to a concentration of 10 mg·mL -1 α-Fe2O3 / Fe3O4@Au nanocomposite suspension was prepared, and then 9 μL was added dropwise to the cleaned MGCE surface and dried at room temperature to obtain the MGCE / α-Fe2O3 / Fe3O4@Au modified electrode.

[0136] MGCE / α-Fe2O3 / Fe3O4@Au / Sub and MGCE / α-Fe2O3 / Fe3O4@Au / Sub / BSA were prepared in Example 2.

[0137] The MGCE / α-Fe2O3 / Fe3O4@Au / Sub / BSA+DNA walker was prepared as in Example 3.

[0138] MGCE / α-Fe2O3 / Fe3O4@Au / Sub / BSA+DNA walker+Cas14a-sgRNA was prepared as in Example 4.

[0139] The test was carried out using a CHI660E electrochemical workstation and a three-electrode system. The bare electrodes MGCE, MGCE / α-Fe2O3 / Fe3O4, MGCE / α-Fe2O3 / Fe3O4@Au, MGCE / α-Fe2O3 / Fe3O4@Au / Sub, MGCE / α-Fe2O3 / Fe3O4@Au / Sub / BSA, MGCE / α-Fe2O3 / Fe3O4@Au / Sub / BSA+DNA walker, and MGCE / α-Fe2O3 / Fe3O4@Au / Sub / BSA+DNA walker+Cas14a-sgRNA were used as working electrodes, Ag / AgCl electrode and Pt electrode were used as reference and auxiliary electrodes, respectively. 5 mM [Fe(CN)6]3- / 4- solution as electrolyte.

[0140] Figure 2 The cyclic voltammetry diagrams for each stage of the electrochemical detection platform construction process under the conditions described in this example show that due to the superior conductivity of the surface AuNPs, the redox current of the α-Fe2O3 / Fe3O4@Au nanocomposite (curve c) is significantly increased compared to the bare electrode curve a and the α-Fe2O3 / Fe3O4 heterogeneous nanoparticles (curve b); this indicates that gold coating helps amplify the signal. After the hairpin reporter subunit Sub is connected (curve d), the current decreases significantly. This is because the negatively charged phosphate backbone of the hairpin DNA reacts with the [Fe(CN)6] 3- / 4- Due to electrostatic repulsion. At the same time, the disordered arrangement of Sub on the surface of the nanocomplex will also lead to an increase in charge transfer resistance. When BSA is used to block the nonspecific binding sites (curve e), the current is further reduced due to the increase in steric hindrance. Subsequently, the constructed bipedal DNA walker cuts the Sub once (curve f), turning the hairpin DNA into two single-stranded DNAs. Compared with the hairpin DNA with a similar rigid structure, the flexible single-stranded DNA will fall on the surface of the nanosensor, thereby hindering the charge transfer and further reducing the current. Finally, the activated Cas14a protein is added to the mixture in the previous step to perform a secondary cutting of the reporter fragments (curve g). Because Cas14a has a trans-cutting effect, the reporter is cut into small fragments like a shredder. Therefore, a mutual attraction is generated between the positively charged nanosensor surface and the negatively charged DNA fragments, which leads to a further increase in the steric hindrance of the signal transmission matrix α-Fe2O3 / Fe3O4@Au surface, further closing the current path, and the [Fe(CN)6] 3- / 4- It becomes more difficult to reach the electrode surface, causing the current value to decrease again.

[0141] In summary, the enzyme-immune electrochemical HPV16 E7 detection platform of the present invention can be used for electrochemical detection of HPV16 E7 oncoprotein.

[0142] Example 9: Selectivity Investigation of the Enzyme-Free Label-Free Electrochemical HPV16 E7 Detection Platform

[0143] In this example, human epidermal growth factor receptor 2 (HER2), plasmin, epithelial cell adhesion molecule (EpCAM), carcinoembryonic antigen (CEA), and a mixture thereof (Mix) were used as interfering proteins to investigate the selectivity of the enzyme-free, label-free, electrochemical HPV16 E7 detection platform. The specific steps are as follows:

[0144] S1. Take 10 μL of the solution and the concentration is 10 ng·mL -1HPV16 E7 and 1000 ng·mL -1 HER2, plasmin, EpCAM, CEA, Mix, and 5 μL of 10 nM A1 were reacted at 37°C for 40 minutes to form a binary complex HA; 5 μL of 10 nM single-stranded DNA D1 was added to the binary complex HA and reacted at 37°C for 40 minutes, after which a ternary complex HAD was formed; then 5 μL of 10 nM single-stranded DNA D2 was added to the ternary complex HAD and a chain displacement reaction was carried out at 37°C for 40 minutes, after which a double-stranded bipedal DNA walker mixture with two single-stranded ends was formed.

[0145] S2. Add 25 μL of the double-stranded bipedal DNA walker mixture with two single-stranded ends to the signal transmission medium α-Fe2O3 / Fe3O4@Au / Sub / BSA obtained in step (1), and then add 5 μL of 25 mM Mg 2+ The solution was mixed, allowed to stand at room temperature for 1 h, and the DNA walking program was started to obtain an enzyme-free bipedal DNA walker.

[0146] S3. Add 20 μL of the binary complex Cas14a-sgRNA prepared in Example 4 to 30 μL of the reaction mixture solution of S2, mix gently, lyse in the dark for 30 min, and finally inactivate at 80°C for 10 min to obtain the electrochemical detection platform.

[0147] Differential pulse voltammetry (DPV) was used to detect the selective current response of the electrochemical detection platform obtained in this embodiment to HPV16 E7 oncoprotein and HER2, plasmin, EpCAM, CEA and Mix. The detection results are as follows: Figure 3 shown.

[0148] Figure 3 This is the selective current response of the electrochemical detection platform to HPV16 E7 oncoprotein and HER2, plasmin, EpCAM, CEA and Mix under the conditions described in this example. Figure 3 It can be seen that when the concentration of the interfering substance is 100 times higher than that of HPV16 E7 oncoprotein, the electrical signal intensity of HPV16 E7 oncoprotein is only 28.02% of that of the remaining interfering substances, indicating that the electrochemical detection platform has good selectivity.

[0149] Example 10: Evaluation of the quantitative analysis capability of the enzyme-free label-free electrochemical HPV16 E7 detection platform

[0150] This example investigates the quantitative analysis capability of the enzyme-free label-free electrochemical HPV16 E7 detection platform, and the specific steps are as follows.

[0151] Take 10uL of different concentrations of HPV16 E7 (10 -2 pg·mL -1 ~10 8 pg·mL -1 ) The bipedal walker preparation process described in Example 3 was carried out, and then 25 μL of the obtained mixture was added to the signal transmission medium α-Fe2O3 / Fe3O4@Au / Sub / BSA prepared in Example 2, and then 5 μL of Mg 2+ The (2.5mM) solution was mixed, allowed to stand at room temperature for 1.5h, and the DNA walking program was started. After the reaction was completed, the binary complex Cas14a-sgRNA prepared in Example 4 was added, gently mixed, lysed in the dark for 30min, and finally inactivated at 80℃ for 10min to obtain the electrochemical detection platform prepared under different concentrations of HPV16 E7. DPV detection was performed on the electrochemical detection platform prepared under different concentrations of HPV16 E7 oncoprotein, and a linear current response was obtained. The detection results are as follows Figure 4 shown.

[0152] Figure 4 This is the linear current response of the electrochemical detection platform to different concentrations of HPV16 E7 oncoprotein under the conditions described in this example. Figure 4 As can be seen in A, as the concentration of HPV16 E7 increases, the peak current gradually decreases, and the change trend is consistent with the principle of this detection platform; Figure 4 B shows that the peak current is log-linearly related to the HPV16 E7 concentration. The concentration that this nanoprobe can detect is 100 fg·mL -1 ~10 μg·mL -1 At 100 fg·mL -1 to 10 ng·mL -1 Within the range, the linear equation is I(μA)=34.44–5.92*lg C HPV16 E7 (pg·mL -1 ), R 2 =0.9998; at 10 ng·mL -1 to 10 μg·mL -1 In the range of , the linear equation is I(μA)=17.91–1.96*lg C HPV16 E7 (pg·mL -1 ), R 2 =0.995. The detection limit was calculated by linear fitting the data to be 67.17 fg·mL -1 (LOQ = 3σ / m), indicating its excellent ultrasensitive detection ability for HPV16 E7 protein.

[0153] In summary, compared with the existing technology, the self-assembled, enzyme-free, label-free, electrochemical HPV16 E7 detection platform preparation method described in the present invention has many advantages: First, the preparation process of this method is simple and easy, and does not require complex experimental operations and expensive equipment. Second, this method has good selectivity, and can accurately identify and capture the target substance HPV16 E7 oncoprotein during the detection process, while eliminating the influence of other interfering factors. In addition, this method also has an extremely low detection limit, capable of achieving highly sensitive detection of HPV16 E7 oncoprotein at very low concentrations. This self-assembled, enzyme-free, label-free, electrochemical HPV16 E7 detection platform preparation method provides the medical field with a rapid, reliable, and cost-effective means for early screening and diagnosis of HPV-related diseases. It can be widely used in clinical practice, helping doctors to promptly identify the risk of HPV infection in patients and further assess whether it will lead to the development of malignant tumors.

[0154] The embodiments described are preferred implementations of the present invention, but the present invention is not limited to the above implementations. Any obvious improvements, substitutions or modifications that can be made by those skilled in the art without departing from the essence of the present invention are within the scope of protection of the present invention.

Claims

1. A self-assembled label-free and enzyme-free electrochemical HPV16 E7 detection platform, characterized in that: The electrochemical HPV16E7 detection platform includes: α-Fe2O3 / Fe3O4@Au / Sub / BSA signal transmission medium, CRISPR / Cas14a signal amplification system and enzyme-free bipedal DNA walker; The magnetic α-Fe2O3 / Fe3O4@Au / Sub / BSA signal transmission medium includes α-Fe2O3 / Fe3O4@Au nanoparticles, a hairpin structure reporter Sub assembled on the α-Fe2O3 / Fe3O4@Au nanoparticles, and a non-specific site blocker bovine serum albumin BSA; The CRISPR / Cas14a signal amplification system is assembled from Cas12a enzyme and sgRNA; The enzyme-free bipedal DNA walker includes HPV16 E7, aptamer A1, Mg 2+ , ssDNA D1 and ssDNA D2 and start the walking program.

2. The self-assembled label-free and enzyme-free electrochemical HPV16 E7 detection platform according to claim 1, characterized in that: The nucleotide sequence of the sgRNA is shown in SEQ ID No. 1; The nucleotide sequence of the hairpin structure reporter Sub is shown in SEQ ID No. 2; The nucleotide sequences of the aptamer A1, single-stranded DNA D1 and single-stranded DNA D2 are shown in SEQ ID No.3 to SEQ ID No.

5.

3. The method for preparing the self-assembled label-free and enzyme-free electrochemical HPV16 E7 detection platform according to claim 1 or 2, characterized in that: The preparation method comprises: (1) Construction of magnetic α-Fe2O3 / Fe3O4@Au / Sub / BSA signal transmission medium: The α-Fe2O3 / Fe3O4@Au nanoparticle suspension was mixed with the hairpin structure reporter Sub, and then bovine serum albumin BSA was added to block the nonspecific adsorption sites to obtain the magnetic α-Fe2O3 / Fe3O4@Au / Sub / BSA signal transmission medium; (2) Construction and activation of enzyme-free bipedal DNA walker: HPV16 E7 and aptamer A1 are mixed and incubated to obtain a binary complex HA, and then single-stranded DNA D1 is added to the binary complex HA. The complementary hybridization between single-stranded DNA D1 and aptamer A1 obtains a ternary complex HAD, and then single-stranded DNA D2 is added thereto to construct a DNA walker through base complementary pairing to obtain a double-stranded bipedal DNA walker mixture with two single-stranded ends; The double-stranded bipedal DNA walker mixture with two single-stranded ends was added to the magnetic α-Fe2O3 / Fe3O4@Au / Sub / BSA signal transmission medium, and Mg was added to it. 2+ The solution mixing reaction is used to start the DNA walker, and a mixture is obtained after the reaction is completed; (3) Preparation of self-assembled label-free and enzyme-free electrochemical HPV16 E7 detection platform: The Cas14a and sgRNA solution are incubated and assembled in HOLMES Buffer 1 to form a Cas14a-sgRNA binary complex; the Cas14a-sgRNA binary complex is added to the mixture obtained in step (2) to perform a cleavage reaction, and after the reaction, the Cas14a enzyme is inactivated to finally obtain the self-assembled label-free enzyme-free electrochemical HPV16 E7 detection platform.

4. The method for preparing the self-assembled label-free and enzyme-free electrochemical HPV16 E7 detection platform according to claim 3, characterized in that: In step (1), the α-Fe2O3 / Fe3O4@Au nanoparticle suspension and the hairpin structure reporter Sub are mixed in equal volumes, wherein the concentration of the α-Fe2O3 / Fe3O4@Au nanoparticle suspension is 1 mg·mL -1 ~40 mg mL -1 ; The concentration of Sub is 0.01μM~50μM; The conditions for blocking the non-specific adsorption sites with bovine serum albumin (BSA) are as follows: adding BSA with a mass concentration of 0.05% to 25% and incubating at 15 to 57° C. for 0.5 to 24 hours.

5. The method for preparing the self-assembled label-free and enzyme-free electrochemical HPV16 E7 detection platform according to claim 3, characterized in that: In step (2), the concentration relationship of the aptamer A1, single-stranded DNA D1 and single-stranded DNA D2 in the system is 1pM-10mM: 1pM-10mM: 1pM-10mM; the volume of HPV16 E7 and the aptamer A1, single-stranded DNA D1 and single-stranded DNA D2 is 2:1:1:1; The conditions for constructing the DNA walker are: reacting at 15-57° C. for 1-60 min.

6. The method for preparing the self-assembled label-free and enzyme-free electrochemical HPV16 E7 detection platform according to claim 3, characterized in that: In step (2), the Mg 2+ The concentration is 0.1mM~100mM, and the time of the mixing reaction initiated by the DNA walker is 0.1~3h.

7. The method for preparing the self-assembled label-free and enzyme-free electrochemical HPV16 E7 detection platform according to claim 3, characterized in that: In step (3), the Cas14a and sgRNA are mixed in equal volumes; wherein the concentration of Cas14a is 10nM to 500nM, and the concentration of sgRNA is 10nM to 500nM. The incubation assembly conditions are: reacting at 15-57° C. for 1-60 min; The cleavage reaction conditions last for 10 to 90 minutes; The conditions of the enzyme inactivation treatment are: reacting at 30-100° C. for 1-60 min.

8. Use of the self-assembled label-free and enzyme-free electrochemical HPV16 E7 detection platform according to any one of claims 1 or 2 in the preparation of an electrochemical sensor for detecting HPV16 E7 protein.

9. An electrochemical sensor for detecting HPV16 E7 protein, characterized in that: The electrochemical sensor uses the self-assembled label-free and enzyme-free electrochemical HPV16 E7 detection platform as the working electrode, the Ag / AgCl electrode and the Pt electrode as the reference and auxiliary electrodes, respectively, and the [Fe(CN)6] 3- / 4- The solution was used as electrolyte, and the concentration of HPV16 E7 protein was detected by differential pulse voltammetry using an electrochemical workstation.

10. The electrochemical sensor for detecting HPV16 E7 protein according to claim 9, characterized in that: In the electrolyte, the concentration of KCl is 0.1M, [Fe(CN)6] 3- / 4- The concentration of the solution is 5 mM; When the electrochemical nucleic acid sensor is used for detection, the potential range of CV is from -0.1V to +0.7V, and the electrochemical impedance spectrum is within the range of 0.1Hz-10kHz, measured with a signal amplitude of 0.1V.

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